Abstract (30 lines of text) The mechanisms by which global regulatory proteins operate on their RNA targets are complex and are still largely not understood; understanding these mechanisms is crucial for preventing or alleviating bacterial infections. As one of the major established global post-transcriptional regulator in bacteria, the CsrA RNA- binding protein hides a wealth of strategies for efficient stress-induced posttranscriptional regulation that are not well understood. The overarching goal of the proposed work is to characterize in vivo the molecular features that underlie novel CsrA-target interactions that we have recently uncovered as well as to decipher the biological relevance of these interactions. The CsrA protein is part of the Csr (carbon storage regulator) system, a global regulatory network known to impact virulence factors, secretion systems, and other important genes for rapid host adaptation in a number of pathogens. Based on our recent discoveries, we propose the new hypotheses that: (i) CsrA has diverse types of binding sites on its targets, (ii) CsrA can have multiple binding site combinations within a single target that can lead to different regulatory outcomes, (iii) CsrA interacts with and contributes to the regulation of a wide range of cellular sRNAs, and that (iv) noncanonical features of CsrA regulation are conserved beyond E. coli. Our overall objective is to test these hypotheses in the context of the E. coli CsrA targetome and of the homologue RsmA targetome in P. aeruginosa. Based on our overall goal, we propose three Specific Aims: (1) to characterize in vivo the regulatory binding sites of the CsrA targetome, (2) to characterize in vivo CsrA-sRNA interactions and their functional relevance, and (3) to characterize RsmA-target binding and regulation in P. aeruginosa. We expect that these studies will expand our current paradigm of post-transcriptional regulation approaches that global regulators exert in bacteria. We also expect that this fundamental work will continue to provide framework for studies of CsrA regulation and of other global RNA-binding regulatory proteins to inform their potential use as therapeutic targets. A highly innovative aspect of this work is the use of new intracellular probing tools developed in our lab.